IMMOBILIZED

ENZYME SYSTEM
CHE142 Lecture 3.1
 Effect of pH and temperature

-Enzyme are active only over small range of pH due to

the active site functional group charges (ionic form) and
the three dimensional shape of enzyme are pH-
dependent
-Certain enzyme have ionic group on their active sites,
and these ionic group must be in a suitable form (acid or
base) to function.
-Variation in pH of medium result in changes of:
-Ionic form of the active site
-Activity of enzyme, hence the reaction rate
-Affect the maximum reaction rate, Km
-stability of enzyme
 -Scheme to describe pH dependence of the enzymatic
reaction rate for ionizing enzymes.
-

Variation of enzyme activity

with pH for 2 different
enzymes

-Some cases, the substrate may contain ionic groups,

and the pH of medium affects the affinity of substrate to
enzyme.
-Refer to Eq. 3.44& Eq. 3.45
 ascending

descending
Thermal
Denaturation
occurred
The rate varies Variation of reaction rate with temperature
according to
Arrhenius equation
Restriction of enzyme mobility
in a fixed space = enzyme
immobilization
Immobilized Enzymes

Immobilization – enzyme has been contained or

localized so it can be reused continuously
■ Immobilized enzymes are enzymes which are attached in or onto the
surface of an insoluble support

• Immobilized enzymes have several advantages over the soluble

enzyme:
– Convenience: Miniscule amounts of protein dissolve in the
reaction, so workup can be much easier. Upon completion,
reaction mixtures typically contain only solvent and reaction
products.
– Economical: easily removed from the reaction  reusage
– Stability: Immobilized enzymes typically have greater thermal
and operational stability than the soluble form of the enzyme
 Immobilization criteria
■ There are a number of requirements to achieve a
successful immobilization:
– The biological component must retain
substantial biological activity after attachment
– It must have a long-term stability
– The sensitivity of the enzyme must be
preserved after attachment
– Overloading can block or inactivate the active
site of the immobilized biomaterial, therefore,
must be avoided
Selecting an Immobilization
Technique

■It is well recognized that no one method

can be regarded as the universal method
for all applications or all enzymes.
Consider,
– widely different chemical
characteristics of enzymes
– different properties of substrates and
products
– range of potential processes employed
Immobilized Enzyme Systems
Enzyme Immobilization:
- Easy separation from reaction mixture, providing the ability
to control reaction times and minimize the enzymes lost
in the product.

- Re-use of enzymes for many reaction cycles, lowering the

total production cost of enzyme mediated reactions.

- Ability of enzymes to provide pure products.

- Possible provision of a better environment for enzyme

activity

- Diffusional limitation
Classification of Immobilization Methods
for Enzymes
Methods of Enzyme Immobilization
Immobilization by Entrapment
Entrapment Immobilization is based on
the localization of an enzyme within
the lattice of a polymer matrix or
membrane.
- retain enzyme
- allow the penetration of substrate.

It can be classified into matrix and

micro capsule types.
 Gel-fibre entrapment and encapsulation
Entrapment
■ Enzymes may be entrapped within the matrix of a polymeric gel
– Incubate the enzyme together with the gel monomers
– Promote gel polymerization
■ Polyacrylamide and polymethacrylamide gels are examples
■ Gel pore size is a crucial factor

Encapsulation
■ Encapsulation involves entrapping the enzymes within a
semipermeable membrane such as cellulose nitrate and nylon-
based membranes
 Immobilized Enzyme Systems
Entrapment
- Matrix Entrapment - Membrane
Entrapment

(microencapsulation)
■Gel entrapment places the enzyme within the
interstitial
■spaces of crosslinked, water-insoluble polymer gels.
■Polyacrylamide gels:
■Polysaccharides: The solubility of alginate and k-
Carrageenan varies with the cation, allowing these
soluble polymers to be crosslinked upon the addition of
CaCl2 and KCl, respectively.

■Variations of pore size result in enzyme leakage, even

after washing. The effect of initiator used in
polyacrylamide gels can be problematic.

cont……….
 Immobilized Enzyme Systems

Matrix Materials:

Organics: polysaccharides, proteins, carbon, vinyl and

allyl polymers, and polyamides. e.g. Ca-alginate, agar,
K-carrageenin, collagen

Immobilization procedures:
Enzyme + polymer solution → polymerization
→ extrusion/shape the particles

Inorganics: activated carbon, porous ceramic.

Shapes: particle, membrane, fiber

Immobilization by Entrapment in microcapsule
■Microencapsulation encloses enzymes within spherical,
■semi-permeable membranes of 1-100 mm diameter.
■Urethane prepolymers, when mixed with an aqueous enzyme
solution crosslink via urea bonds to generate membranes of
varying hydrophilicity.
■Alternatively, photo-crosslinkable resins can be gelled by UV-
irradiation.
■Advantage of Entrapment
– Enzymes are immobilized without a chemical or structural
modification. A very general technique.
Disadvantage of Entrapment
– High molecular weight substrates have limited diffusivity,
and cannot be treated with entrapped enzymes.
Matrix Materials used in Entrapment :
Organics: polysaccharides, proteins, carbon, vinyl and
allyl polymers, and polyamides. e.g. Ca-alginate, agar,
K-carrageenin, collagen
Immobilization procedures:
Enzyme + polymer solution → polymerization
→ extrusion/shape the particles
Inorganics: activated carbon, porous ceramic.
Shapes: particle, membrane, fiber
 Immobilized Enzyme Systems
Entrapment
challenges:
- enzyme leakage into solution
- diffusional limitation
- reduced enzyme activity and stability
- lack of control micro-environmental
conditions.
It could be improved by modifying matrix or
membrane.
Allowing small MW
compound access to Retain high MW
enzyme compound
Challenges in Entrapment Method
- enzyme leakage into solution
- diffusional limitation
- reduced enzyme activity and stability
- lack of control micro-environmental
conditions.
It could be improved by modifying matrix or
membrane.
 Immobilization by Carrier Binding or
Surface Immobilization
■Attachment of an enzyme to an insoluble carrier creates an active
surface catalyst. Modes of surface attachment classify carrier
methods into physical adsorption, ionic binding and covalent binding.
■Physical Adsorption: Enzymes can be bound to carriers
■by physical interaction such as hydrogen bonding and/or
■ van der Waal’s forces.
– the enzyme structure is unmodified
– carriers include chitosan, acrylamide polymers and silica-
alumina
– binding strength is usually weak and affected by temperature
and the concentration of reactants.
■Ionic Binding: Stronger enzyme-carrier binding is obtained with solid
supports containing ion-exchange residues.
– cellulose, glass-fibre paper, polystyrene sulfonate
– pH and ionic strength effects can be significant
 Adsorption and Ionic binding
■ Simplest immobilization method
– Mix the enzyme and support in suitable conditions
■ First immobilized enzyme model: invertase on the activated
charcoal (Nelson and Griffin, 1916)
■ Forces are weak so leakage is generally a problem
■ Supports such as alluminium hydroxide are often utilized
■ With a suitable charged matrix, ionic interactions may also
be promoted
■ This technique is technically undemanding and
economically attractive
■ Regeneration is easy
■ Best known industrial example: amino acylase immobilized
on DEAE-Sephadex in the production of amino acids
Surface immobilization
According to the binding mode of the
enzyme, this method can be further sub-
classified into:
- Physical Adsorption: Van der Waals
Carriers: silica, carbon nanotube, cellulose,
etc.
Easily desorbed, simple and cheap,
enzyme activity unaffected.
- Ionic Binding: ionic bonds
Similar to physical adsorption.
Carriers: polysaccharides and synthetic
polymers
having ion-exchange centers.
 Immobilized Enzyme Systems
Surface immobilization
- Covalent Binding: covalent bonds
Carriers: polymers contain amino, carboxyl, sulfhydryl,
hydroxyl, or phenolic groups.

- Loss of enzyme activity

- Strong binding of enzymes
■Covalent attachment of soluble enzymes to an insoluble
support is the most common immobilization technique.
– Amino acid residues not involved in the active site can be
used fix the enzyme to a solid carrier
■Advantages:
1. Minimal enzyme leaching from the support
results in stable productivity
2. Surface placement permits enzyme contact
with large substrates
■Disadvantages:
1. Partial modification of residues that constitute the active
site decreases activity
2. Immobilization conditions can be difficult to optimize (often
done in the presence of a competitive inhibitor)
 Covalent immobilization
■ The most widely used method for enzyme immobilization
– It is technically more complex
– It requires a variety of often expensive chemicals
– It is time-consuming
 But immobilized enzyme preparations are stable and
leaching is minimal
■ Enzymes are immobilized by a suitable group in the
surface:
– Hydroxyl groups in supports (e.g cellulose, dextran,
agarose)
– Amino, carboxyl and sulfhydryl groups in amino
acids
 Covalent immobilization
■ The conditions for immobilization by covalent binding are much more
complicated and less mild than in the cases of physical adsorption
and ionic binding. Therefore, covalent binding may alter the
conformational structure and active center of the enzyme, resulting
in major loss of activity and/or changes of the substrate
■ Covalent attachment to a support matrix must involve only functional
groups of the enzyme that are not essential for catalytic action
■ Higher activities result from prevention of inactivation reactions with
amino acid residues of the active sites. A number of protective
methods have been devised:
– Covalent attachment of the enzyme in the presence of a
competitive inhibitor or substrate
– A chemically modified soluble enzyme whose covalent linkage to
the matrix is achieved by newly incorporated residues
Site-specific immobilization

Three different approaches:

(a) Gene fusion to incorporate a
peptidic affinity tag at the N- or
C-terminus of the enzyme. The
enzymes are then attached
from this affinity tag to anti-tag
antibodies on membranes
(b) Modification to incorporate a
single biotin moiety on enzymes
(see figure)
(c) Site-directed mutagenesis to
introduce unique cysteines to
enzymes. The enzymes are
attached on thiol-reactive
surfaces through the sulfhydryl
group
Prior to
Functional groups on support material are usually
activated by using chemical reagent such as
cyanogen bromide, carbodiimide and
glutaraldehyde
 Properties of support material
■ The form, shape, density, porosity, pore size distribution, operational
stability and particle size distribution of the supporting matrix will
influence the result
■ The ideal support is cheap, inert, physically strong and stable
■ Ideally, it should:
– increase the enzyme specificity (kcat/Km)
– shift the pH optimum to the desired value for the process
– discourage microbial growth and non-specific adsorption
■ Some matrices may possess other properties which are useful for
particular purposes such as
– ferromagnetism (e.g. magnetic iron oxide, enabling transfer of the
biocatalyst by means of magnetic fields)
– a catalytic surface (e.g. manganese dioxide, which catalytically removes
the inactivating hydrogen peroxide produced by most oxidases)
 Chemical reagent
Support
materials
with
functional
group
 Most Convenient Residues
for Covalent Binding
•Amino acid residues with polar and reactive functional
groups are best for covalent binding, given that they
are most often found on the surface of the enzyme.

•The data shown in next slide is the most convenient

residues for binding in descending order.

•The average percent composition of proteins

(reactive residues only) is shown, along with the
number of potential binding reactions in which the
amino acids partake.
 ■Abundance(%)Reactions

■ 7.0 27
+ Lysine (Lys)
CH2 4 NH3
■ 3.4 31
Cysteine (Cys)
CH2 SH
■ 3.4 16

CH2 OH Tyrosine (Tyr)

■ 2.2 13
HN N
Histidine (His)
CH2
O ■ 4.8 4

CH2 C O Aspartic Acid (Asp)

O ■ 4.8 4
CH2 CH2 C O Glutamic Acid (Glu)
■ 3.8 6

CH2 3 NH C NH2 Arginine (Arg)

+NH2
CH2 ■ 1.2 7
Tryptophan (Trp)
N
H
Covalent Attachment Techniques
■Cyanogen bromide activates supports with vicinal hydroxyl groups
(polysaccharides, glass beads) to yield reactive imidocarbonate derivatives:

■Diazonium derivatives of supports having aromatic amino groups are

activated for enzyme immobilization:

■Under the action of condensing agents (Woodward’s reagent K), carboxyl or

amino groups of supports and amino acid residues can be condensed to yield
peptide linkages.
■Other methods include diazo coupling, alkylation, etc.
 Immobilization by Crosslinking
■Bi- or multi-functional compounds serve as reagents
for intermolecular crosslinking of enzymes, creating
insoluble aggregates that are effective
heterogeneous catalysts.

■Reagents commonly have two identical functional

groups which react with specific amino acid residues.

■Common reagents include glutaraldehyde,

carbodimide and diisocyanates,

■Involvement of the active site in crosslinking can

lead to great reductions in activity, and the gelatinous
nature of the product can complicate processing.
Cross-linking is to cross link enzyme
molecules with each other using agents
such as glutaraldehyde.
Features: similar to covalent binding.

Several methods are combined.

 Immobilized Enzymes
■ Advantages
■ Retention in reactor
■ Disadvantages
■ Separation from reaction ■ Mass-transfer
components is facilitated limitations
■ Usable in a wide range of ■ Loss of activity
reactor configurations upon
immobilization
■ High catalytic loadings
■ Impractical for
■ Enhanced stability toward solid substrates
T, pH, solvent, etc.
■ Modified selectivities
Application of Immobilized-
Enzymes

1-High-fructose corn syrups (HFCS)

2-GLUCOSE ISOMERASE
a Treatment with activated carbon.
3-Use of immobilised raffinase
4-Use of immobilised Invertase
5-Production of amino acids
6- Use of immobilised lactase
7- Production of antibiotics
 Effect of Immobilization on
Operational Stability
■Given that activity of enzymes is dictated by structure and
conformation, the environmental change resulting from
immobilization affects not only maximum activity, but the
stability of the enzyme preparation.
– The factors that inactivate enzymes are not
systematically understood, and depend on the intrinsic
nature of the enzyme, the method of immobilization,
and the reaction conditions employed.
– In general, immobilized enzyme preparations
demonstrate better stability
Note that the immobilized preparation is ften more stable than the soluble
enzyme and displays a period during which no enzyme activity appears to be
lost.

immobilized
enzymes

free (soluble)
enzymes
 Effects of Immobilization on Enzyme
Stability and Use
■Design of enzymatic processes requires knowledge of:
– reactant and product selectivity
– thermodynamic equilibria that may limit product yield
– reaction rate as a function of process conditions ([Enzyme], [substrate(s)],
[Inhibitors], temperature, pH, …)

■Two design issues that we have not considered are:

– enzyme stability
– efficiency losses associated with the use of homogeneous (soluble) catalysts

■Immobilization of an enzyme allows

■it to be retained in a continuous reactor,
■but its initial activity and its stability
■directly influence its usefulness
■in industrial applications.
Effects of Enzyme
Immobilization on Activity
Enzyme Stability
■Although enzyme storage stability is important, it is the operational
stability of an enzyme that governs its reactor performance.
– Operation stability is a complex function of temperature, pH, [substrate]
and the presence of destabilizing agents.
■Generally, the rate of free enzyme deactivation is first order with a
deactivation constant, kd:

d[E]T
 k d [E]T
dt
■Integrating this expression yields the concentration of active enzyme
as a function of time:

[E]T  [E]T,o e k dt
Yields of the concentration of active
enzyme as a function of time:
6.0

5.0
[Enzyme] *1E6 M

No decay
4.0
kd = 6E-6 s-1
3.0 kd = 3E-5 s-1

2.0

1.0

0.0
0 20 40 60 80 100
Time (hours)
 Effect of Thermolysin Instability on APM Production
■Recall the rate expression developed for APM synthesis by
thermolysin:
d[ ZAPM] k 2 [E]T [ ZLAsp][LPM]

dt K1  [ ZLAsp]

■If thermolysin deactivation were adequately described as a

first order process, the observed reaction rate would have an
explicit time dependence, as shown below:

d[ ZAPM] k 2 [ ZLAsp][LPM]
 [E]T,o e k dt
dt K1  [ ZLAsp]
■where [E]T,o represents the initial enzyme concentration and kd
is the deactivation rate constant.
■The conversion versus time profile for aspartame synthesis by
a batch process can be developed from this expression by
integration.
 Effect of Thermolysin Instability on APM
Production
■The evolution of [L-Asp] and conversion with
time for a batch process is shown below.
– Depending on the relative rates of reaction
and enzyme deactivation, the ultimate
conversion can be strongly affected
 APM Synthesis by Thermolysin APM Synthesis by Thermolysin
Batch Process at 40C Batch Process at 40C
0.020 0.90
0.018 kd = 0 s-1
0.80
0.016 0.70

L-Asp Conversion
kd = 3E-5 s-1
0.014 kd = 6E-6 s-1
0.60
[L-Asp]: M

0.012
0.50
0.010
kd = 6E-6 s-1 0.40
0.008
0.30 kd = 3E-5 s-1
0.006 kd = 0 s-1
0.004 0.20
0.002 0.10
0.000 0.00
0 20 40 60 80 100 0 20 40 60 80 100
Time (hours) Time (hours)
[LPM]o 0.0182 M [LAsp]o 0.0182 M [LPM]o 0.0182 M [LAsp]o 0.0182 M
k2 2.65 M-1s-1 K1 0.0103 M-1s-1 k2 2.65 M-1s-1 K1 0.0103 M-1s-1
[E]o 4.85E-06 M [E]o 4.85E-06 M
Industrial Enzymatic Synthesis of Aspartame
■The unique regio and stereoselectivity afforded by enzymes has been
exploited on an industrial scale Aspartame production.

CO2H Ph
■The process employs a protease,
NH
■thermolysin, to catalyze the H2N
H CO2Me
H
O
■condensation of the modified Asp
-L-aspartyl-L-phenylanaline methyl ester
■and Phe). -aspartame (APM)]

■The forward reaction is written as:

CO2H
Ph
CO2H Ph
X
N
H H
CO2H + H2N
H CO2Me
thermolysin
X NH + OH2
N
HH H CO2Me
Amine-protected (X) Methyl ester of
L-phenylanaline O
L-aspartic acid
(Z-L-Asp) (L-PM) (APM)
Factors Affecting Immobilized Enzyme
Kinetics
■ pH effects
- on enzymes
- enzymes have ionic groups on their active sites.
- Variation of pH changes the ionic form of the active
sites.
- pH changes the three-Dimensional structure of
enzymes.

- on substrate
- some substrates contain ionic groups
- pH affects the ionic form of substrate
affects the affinity of the substrate to the enzyme.
• Effect of Temperature
- on the rate of enzyme catalyzed reaction
d[ P]
v  k 2 [ ES ]
dt
k2=A*exp(-Ea/R*T)

T k2 v
- enzyme denaturation d[ E ]
  kd [E]
T Denaturation rate: dt
kd =Ad*exp(-Ea/R*T)
Where kd: enzyme denaturation rate constant;
Ea: deactivation energy